Background As they migrate through the developing gut, a sub-population of enteric neural crest-derived cells (ENCCs) begins to differentiate into neurons. The early appearance of neurons raises the possibility that electrical activity and neurotransmitter release could influence the migration or differentiation of ENNCs. Methods The appearance of neuronal sub-types in the gut of embryonic mice was examined using immunohistochemistry. The effects of blocking various forms of neural activity on ENCC migration and neuronal differentiation were examined using explants of cultured embryonic gut. Key Results Nerve fibers were present in close apposition to many ENCCs. Commencing at E11.5, neuronal nitric oxide synthase (nNOS), calbindin and IK Ca channel immunoreactivities were shown by sub-populations of enteric neurons. In cultured explants of embryonic gut, tetrodotoxin (TTX, an inhibitor of action potential generation), nitro-L-arginine (NOLA, an inhibitor of nitric oxide synthesis) and clotrimazole (an IK Ca channel blocker) did not affect the rate of ENCC migration, but tetanus toxin (an inhibitor of SNAREmediated vesicle fusion) significantly impaired ENCC migration as previously reported. In explants of E11.5 and E12.5 hindgut grown in the presence of TTX or tetanus toxin there was a decrease in the number nNOS+ neurons close to the migratory wavefront, but no significant difference in the proportion of all ENCC that expressed the pan-neuronal marker, Hu. Conclusions & Inferences (i) Some enteric neuron sub-types are present very early during the development of the enteric nervous system. (ii) The rate of differentiation of some sub-types of enteric neurons appears to be influenced by TTX-and tetanus toxin-sensitive mechanisms.